Transfection, or the introduction of a DNA molecule into a eukaryotic cell, usually followed by the expression of one or more genes in the newly introduced DNA, represents one of the most important steps in genomics research and gene therapy. While methods for isolating DNA for transfection have improved significantly, effective methods for transfecting isolated DNA strands, especially in in vivo systems, are the limiting factor for progress in gene therapy. A number of transfection methods currently exist, yet each one of them is limited in the scope of its application and each presents certain disadvantages.
Current transfection methods include calcium phosphate precipitation, the use of a cationic lipid--DNA complex, electroporation and the use of viral vectors. Yet, calcium phosphate precipitation does not always yield high levels of transfection in cells. Cationic lipids, used in a complex, are often toxic to cells and thus ineffective for in vivo tranfection for gene therapy. Electroporation is a method where very high voltage levels are used to transport DNA into cells. Since DNA is highly negatively charged, the application of such an electric current allows for the passage of DNA into cells. Yet, this method cannot be used for in vivo transfection. Also, at high voltage levels the death rate of cells is significantly higher, even further limiting the scope of this method.
In viral vector transfection, the DNA to be tranfected is first introduced into the DNA of a virus. The virus, in turn, then injects its DNA, including the desired tranfection DNA, into a host cell. Although this method can be used in in vivo systems, one of its main disadvantages is that the virus can transform itself or its DNA and thus create undesirable side effects such as harmful infection of the host or undesired transformations to host DNA. The utility of this method is thus also significantly limited for gene therapy.
Since current transfection methods are so limited in their scope and utility there is strong need for a non-toxic method for in vivo transfection that has high success rates for transporting DNA into cells and that minimizes harmful side effects. Also, since the specificity of current methods is very limited, a more specific method for transfection is needed to ensure that desired DNA fragments are introduced into specific target cells. The present invention describes a mucin-DNA complex which represents a novel and highly effective method for transfection.
Mucins are glycoproteins with a very high molecular weight (usually more than 1 million Daltons). Mucins are generally about 60 percent or more carbohydrate by composition and water soluble. The carbohydrate molecules are generally attached as chains to the backbone of the proteins. Since carbohydrates are generally linear molecules the resulting structure can be likened to that of a comb, with the carbohydrate molecules forming individual prongs. When such a mucin molecule is combined with isolated strands of DNA a complex is formed in which the carbohydrate and protein molecules of mucin entangle the DNA strands to form a mucin-DNA complex. Said mucin-DNA complex can be precipitated using a number of different methods. Said complex can also be re-suspended and centrifuged to extract desired components of the complex.